V1-vasopressin antagonists

ABSTRACT

Vasopressin derivatives having V 1  and oxytocin antagonist activity whose structures are characterized by a Mpa unit at position 1 and a des-Pro unit at position 7 are prepared by standard peptide synthetic methods also using an oxidative cyclization of a dimercaptan. Representative species are [1-deaminopenicillamine-2-(O-methyl)-tyrosine-7-desproline-8-arginine-9-desglycine]vasopressin or [1-β-mercaptopropionic acid-2-D-(O-ethyl)tyrosine-3-isoleucine-4-threonine-7-desproline-8-arginine-9-desglycine]-vasopressin.

This invention relates to cyclic peptides which have vasopressin (VSP)antagonist activity. More specifically, these new chemical compoundshave VSP or OXT-like structures which are characterized by (1) the lackof a prolyl unit at position 7 and (2) an acyclic β-mercaptopropionicacid residue at position 1. Representative compounds of this new genericgroup have demonstrated weak V₂ -antagonistic activity while maintainingV₁ -antagonistic activity in good degree.

BACKGROUND OF THE INVENTION

A number of synthetic modifications of the vasopressin and oxytocinstructures have been reported to give antagonistic activities. Suchstructures contain units which are derived fromβ-mercapto-β,β-dialkylpropionic acid, for example, deamino-penicillanicacid or β-mercaptopropionic acid, substituted for the cysteine unit atposition 1 of the structure of the natural product: J. Lowbridge et al.,J. Med. Chem. 22 565 (1979); M. Manning et al., J. Med. Chem. 20 1228(1977); K. Bankowski et al., J. Med. Chem. 21 350 (1978); H. Schulz etal., J. Med. Chem. 9 647 (1966).

Ferring, A. B., European Pat. No. 112,809-A, discloses that certainoxytocin compounds with Mpr at position 1 have anti-OXT activity.

Later studies by M. Manning et al., J. Med. Chem. 25 (1982) andPeptides: Chemistry, Structure and Biology (Ann Arbor Sciences) 737(1975), demonstrated that no clearly consistent pattern of increasing ordecreasing antagonist potency has emerged but, in most of the seriesstudied, the β,β-diethyl and β,β-cyclopentamethylene propionic acidunits at position 1 were more active than were the lower homologues, seecolumn 1 on page 411 of the first reference.

We have previously found that removing the proline unit from the tailunits, at position 7, on 1-Pmp-VSP structures gave compounds whichretained the VSP antagonist activity of the parent compounds, F. Ali etal., Ser. Nos. 586,933 and 586,934, both filed on Mar. 7, 1984 and nowissued, U.S. Pat. Nos. 4,481,193 and 4,481,194, respectively. Now, wehave found that removing the proline from a 1-dPen or Mpr-VSP derivativegives strong V₁ -antagonism with a shift in the V₁ :V₂ ratio to theformer.

In the description herein and in the claims, the nomenclature common inthe art of peptide and, more specifically, vasopressin chemistry isused. When no configuration is noted, the amino acid unit is in the L,or naturally occuring, form. The thio members of the β-mercaptopropionicacid (1) and cysteine (6) units are added for clarity in certainstructural formulas.

Exemplary of the peptide art designations used herein are the following:dPen, β-mercapto-β-β-dimethylpropionic acid; Mpr, β-mercaptopropionicacid; Thr, threonine; Orn, ornithine; OXT, oxytocin; Abu, α-aminobutyricacid; Chg, cyclohexylglycine; Cha, cyclohexylalanine; Pba,α-aminophenylbutyric acid; Gln, glutamine; Gly, glycine; Tyr, tyrosine;Phe, phenylalanine; Val, valine; Ile, isoleucine; Nle, norleucine; Leu,leucine; Ala, alanine; Lys, lysine; Arg, arginine; Harg, homoarginine,Asn, asparagine; Tos, tosylate; BHA, benzhydrylamine; DIEA,diisopropylethylamine; 4-MeBzl, 4-methylbenzyl; TFA, trifluoroaceticacid; DCC, dicyclohexylcarbodiimide; HBT, 1-hydroxybenzotriazole; ACM,acetamidomethyl; Mpa, generic β-mercaptopropionic acids.

DESCRIPTION OF THE INVENTION

The des-Pro-vasopressin-like compounds of the invention are illustratedby the following structural formula: ##STR1## in which: P is Phe, Ile,Phe(4'--Alk), Tyr or Tyr(Alk);

X is a D or L isomer of Val, Nva, Leu, Ile, Pba, Phe, Phe(4'--Alk), Trp,Nle, Cha, Abu, Met, Chg, Tyr or Tyr(Alk);

Y is Val, Ile, Abu, Ala, Chg, Gln, Lys, Cha, Thr, Nle, Phe, Leu or Gly;

Z is a D or L isomer of Arg, Harg, Leu, Lys or Orn;

A is Gly(NH₂), Gly, Gly(NH--Alk), OH, NH₂ or NHAlk; and

R is, each, hydrogen or methyl; or a pharmaceutically acceptable salt,prodrug ester or complex thereof.

"Alk" in formula 1 and hereafter represents a lower alkyl of 1-4 carbonswhich may be a substituent which is optionally attached either to theamide nitrogen at A, to a phenyl of an amino acid unit such as Phe atposition 2 or 3 or to the oxygen substituent such as that of a tyrosineunit when the latter is present at position 2. Such alkyl substituentsinclude methyl, ethyl, n-propyl, isopropyl or butyl. Preferably, Alk ismethyl or ethyl. "Bzl" represents benzyl.

When the term, "vasopressin" or "VSP", is used, it means L-argininevasopressin (AVP) unless otherwise modified to indicate a D-arginine,leucine, homoarginine, lysine or ornithine-containing vasopressin.Certain antagonists which have structures related to oxytocin (OXT) arealso included in this invention.

In the compounds represented by formula I, those with structures havingdPen at position 1, a L unit at 2 and an arginine at 7 are preferred forselective VSP V₁ -antagonism, which is manifested by vasodilation.

The β-mercaptopropionic acid unit at position 1, Mpa¹, includes theβ,β-dimethyl congener as well as the β-methyl congener in the form ofone of its separated isomers or a mixture of isomers.

A subgeneric group of compounds of this invention comprises compounds offormula I in which P is Phe, X is Tyr or Tyr(Alk); Y is Ile or Gln; A isGlyNH₂ or NH₂ ; each R is methyl and Z is Arg, Harg, D-Arg, Orn or Leu.

Also included in this invention are various derivatives of the compoundsof formula I such as addition salts, prodrugs in ester or amide form andcomplexes. The addition salts may be either salts with pharmaceuticallyacceptable cations such as NH₄.sup.⊕, Ca.sup.⊕⊕, K.sup.⊕ or Na.sup.⊕ ata terminal acid group, when present, or with a pharmaceuticallyacceptable salt at a basic center of the peptide (as in the Arg or Hargunits). The acetate salt forms are especially useful althoughhydrochloride, hydrobromide and salts with other strong acids areuseful. In the isolation procedures outlined in the Examples, thepeptide product is often isolated as the acetate salt. The compoundsalso form inner salts or zwitter ions as when a free terminal carboxygroup is present.

Prodrugs are derivatives of the compounds of formula I which degrade tothe parent compound in vivo. The ester prodrug forms are, for example,lower alkyl esters of the acids of formula I which have from 1-8 carbonsin the alkyl radical or aralkyl esters which have 6-12 carbons in thearalkyl radical such as various benzyl esters. Other latentiatedderivatives of the compounds of formula I will be obvious to thoseskilled in the art. "Complexes" include various solvates, such ashydrates or alcoholates, or those with supporting resins, such as aMerrifield resin.

The compounds of formula I are prepared by cyclizing a linear peptideintermediate of this invention by means of the two mercapto groupslocated, respectively, in the cysteine unit at position 6 and in theβ-mercaptopropionic acid unit at position 1. The cyclization reactionoccurs in the presence of a mild oxidizing agent which, at highdilution, is capable of oxidizing intramolecularly the dimercaptan to adisulfide.

Oxidation of the following linear peptide; ##STR2##

In which R, P, X, Y, Z, and A are as defined for formula I, but also inwhich Z is a single bond when A is Gly or OH, is carried out asdescribed generally above. For example, an excess of an alkali metalferricyanide, such as potassium or sodium ferricyanide, is used. Thelinear intermediate is dissolved in a suitable unreactive solvent,preferably in an aqueous solvent, at a neutral pH, about 7-7.5. Thereaction is run at ambient temperature, or lower, until substantiallycomplete. Lower alcohols, such as methanol, may be added. Preferably,the concentrations of the linear peptide dimercaptan and the oxidizingagent are low, say 0.01 molar concentration of oxidizing agent inseveral liters of aqueous solution to cyclize 1-6 grams of dimercaptan.

Other mild oxidation agents having an oxidation potential roughlyequivalent to ferricyanide may also be used for the ring closurereaction. Oxygen or iodine are alternatives. Of course, one skilled inthe art will recognize that certain cyclization methods are notappropriate if an interfering reaction site is present in the structureof the starting material of formula II. The linear mercaptan startingmaterial may or may not have protecting groups common the art present atthe various amino acid units or at the mercapto positions. In the formercase, the protecting groups are removed after cyclization. In the caseof the ACM-SH protecting groups, removal of the protective group andcyclization may both be accomplished using iodine in aqueous methanol.Usually, however, the free linear peptide is cyclized.

The desired cyclic des-proline peptides of formula I are convenientlyisolated by acidifying the aqueous oxidation mixture, such as usingglacial acetic acid, and passing the reaction mixture over anion-exchange chromatographic column, for example, over a weakly acid,acrylic resin column with acid elution, or by gel filtration over abead-formed gel prepared by cross-linking dextran with epichlorohydrin.Often, the acetate salt is isolated by this method.

In an alternative reaction sequence for preparing the compounds of thisinvention, the intermediate of formula II in which one or both tailunits is missing is cyclized as described above. The cyclized productis, then, condensed in one or two optional reactions with the protectedamino acid or dipeptide units, which are defined as Z and A for formulaI, to extend the tail portion of the structure. Reaction conditions forsuch tail unit attachment are those of any amide producing method knownto the peptide art as described herein. Particularly, reaction of thetail amino acids, whose carboxylic acid group is protected as described,with the 6-Cys acid in the presence of dicyclohexylcarbodiimide and HBTis used. Thd protecting groups which may be present on the cyclic Cysacid or the tail units are then removed to give the products of thisinvention. Reaction conditions should be selected to minimizeracemization of the Cys unit as known to the art.

The important intermediates of formula II, in free or protected form areconveniently prepared using solid-phase methods of peptide synthesis asdiscussed in M. Manning et al., J. Med. Chem. 25 46 (1982). A commercialbenzhydrylamine support resin (BHR) is used to prepare the amide endproducts of formula I, i.e. in which A is Gly(NH₂), (the amides) and achloromethyl support resin (CMR) is used to prepare the acid compoundsof formula I, i.e. in which A is Gly, (the acids). Solution or enzymaticsynthetic methods can also be used.

The peptide chain of the linear peptides of formula II is built up,stepwise, proceeding from unit 7 or 8 working toward the characterizingunit 1. Each unit is properly protected as known in the peptide art andas described below. The sequence of step reactions is convenientlycarried out in a Beckman 990-B peptide synthesizer without isolation ofeach intermediate peptide. The details of the overall syntheticprocedure are in the working examples presented hereinafter.

The various amino acids, which are consecutively added to the resinsupported chain are protected as known to the art. For example, the Bocprotecting group is used for an amino group especially at the α-positionof the amino acid; ethylcarbamoyl, adamantyl, t-butyl, acetamidomethyl,trityl or an optionally substituted benzyl, for the mercapto groups atthe propionic acid and Cys units; nitro; carbobenzoxy,methylene-2-sulfonyl or tosyl for the Arg unit; and ethyloxycarbonyl oran optionally substituted carbobenzoxy(Z) for the Orn, Tyr or Lys units.The protective groups should, most conveniently, be those which areeasily removed, such as, using acid treatment for thetert.-butyloxycarbonyl (Boc) group, sodium-liquid ammonia or modifiedcatalytic hydrogenation for the benzyl or carbobenzoxy groups.

The protected linear peptide intermediate is split from the carryingresin matrix, for example, by using ammonia in an aqueous misciblesolvent, and, then, is treated to remove the protective groups, such asby using sodium-liquid ammonia. This procedure gives the amidederivative of the linear hepta- or octapeptide.

More conveniently, the two steps are combined by treating the resinsupported peptide with anhydrous hydrogen fluoride using a suitablecarbonium ion scavenger, such as anisole, to give the des-proline dPen¹or Mpr¹ peptide intermediate of formula II in good yield.

The compounds of this invention have potent V₁ -V₂ vasopressinantagonist activity with a shift toward the V₁ -receptors. Vasopressinis primarily known to contribute to the anti-diuretic mechanism ofaction within the kidney. When the action of these compounds antagonizesthat of the natural anti-diuretic hormone (ADH), the body excretes waterdue to an increased permeability of the terminal portions of the renaltubule. This mechanism of action is at the vasopressin receptors (V₂-receptors) located on the plasma membrane of certain renal epithelialcells.

Any patient suffering from the syndrome of inappropriate antidiuretichormone secretion (SIADH) or from an undesirable edematous condition isa target for compounds which have substantial V₂ -antagonist activity.Examples of clinical conditions indicated for such compounds includehypertension, hepatic cirrhosis, congestive heart failure or a componentof any traumatic condition resulting from serious injury or disease.

The second group of vasopressin receptors, which are more importanttargets of this invention, are those affecting the smooth muscle tissuesof the blood vessels or of the uterus. Vasopressin or oxytocin arenatural stimulants of these effectors which result in pressor effects onthe cardiovascular system and stimulation of the uterus, respectively.These receptors are called generically V₁ receptors for the purposes ofthis disclosure. The compounds of the present invention antagonize theactivity of vasopressin and oxytocin at their receptor sites discussedabove.

The compounds of the present invention are, therefore, antagonists at V₁receptor sites. In fact, the V₁ -V₂ ratio of activities of the compoundsof this invention is shifted to give potent V₁ antagonism with very weakV₂ antagonism. Especially active V₁ antagonists are the des-Pro-dPencompounds of formula I in which X is a tyrosyl residue and Y is aglutamine residue.

These compounds, therefore, have selective vasodilation activity whichis of benefit in dilating arterial systems in which vasopressin is amediator. The pharmacological activity is manifested in treatinghypertension, shock or cardiac insufficiency. They are used inconjunction with ACE inhibitors, α-blockers or β-blockers.

The compounds of formula I which have amino acid units other than atposition 1 which resemble those of oxytocin have especially potentanti-oxytocic activity. Therefore, such compounds are particularlyuseful to relax uterine tissues or dry up milk production. Exemplary ofsuch compounds are the des-Pro-Mpr compounds of formula I in which Z isarginine or ornithine; X is a D-Tyr(alk); P is isoleucine; and Y isglutamine or threonine.

The compounds of this invention, therefore, are mainly used to induceselective vasodilation as noted above or to induce antioxytocic activityin patients in need of such antagonist treatment by administrationinternally, particularly parenterally or by insufflation, to saidpatients. A nontoxic but effective quantity of the chosen compound ispreferably combined with a pharmaceutical carrier. Dosage units containa nontoxic, effective quantity of the active ingredient selected fromthe range 0.05-50 mcg/kg, preferably 1-15 mcg/kg, based on a 70 kgpatient. The dosage units are administered from 1 to 5 times daily or bycontinuous intravenous drip.

The pharmaceutical compositions of this invention, which contain anactive ingredient of formula I, comprise a dosage unit quantity asdescribed above dissolved or suspended in a standard liquid carrier.Such a carrier is isotonic saline. The composition is often used in anampoule or a multiple dose vial suitable for parenteral injection, suchas for intravenous, subcutaneous or intramuscular administration. Acomposition for insufflation may be similar but is often administered ina metered dose applicator or inhaler. Pulverized powder compositions maybe used along with oily preparations, gels, buffers for isotonicpreparations, emulsions or aerosols.

Antagonistic activity at the V₁ -vasopressin receptors is determined ina protocol which measures the reversal of the vasopressin-inducedcontraction of rat thoracic aorta tissue. This is expressed as K_(B)(nM) in the table below. Such anti-pressor activity is confirmed in asimilar in vitro protocol using the plasma membranes of rat liver. V₂-vasopressin antagonism is determined as receptor binding abilitymeasured by inhibition of 3H-LVP binding (K_(B) as nM), by inhibition ofadenylate cyclase activation by vasopressin in the medullary tissue ofhog kidneys (Ki as nM) or in vivo in the hydropenic rat protocol (ED₃₀₀μg/kg). These procedures are described in the literature: F. Stassen etal., J. Pharmacology and Experimental Therapeutics, 223, 50 (1982); F.Stassen et al., 1st International Conference on Diuretics, Miami,Florida, March (1984). Antagonistic activity at oxytocin receptors isdetermined in the isolated rat uterus protocol: W. Sawyer, et al.,Endocrinology 106 81 (1979); P. Melin, et al., J. of Endocrinology 88173 (1981).

                                      TABLE 1                                     __________________________________________________________________________    Representative Antagonist Activity                                            Pig V.sub.2.sup.a                                                                             Rat V.sub.1.sup.b                                                                  Rat V.sub.1.sup.c                                                                  Rat OXT.sup.d                                                                       Rat V.sub.2.sup.e                             Compound                                                                            Ki (nM)                                                                            K.sub.B (nM)                                                                       K.sub.B (nM)                                                                       K.sub.B (nM)                                                                       K.sub.B (nM)                                                                        ED.sub.300 (μg/kg)                         __________________________________________________________________________    A     --   3000 38   5.69 32.6  >5959                                         B     --   3300 37   1.48 18.9  >5977                                         C     --    350 --   0.0072.sup.f                                                                       1.88  --                                            D     --   1600 --   --   11.4  ˜2037                                   E     --   4100 --   --   17.7  >5018                                         F     agonist                                                                             70  2.9  --   --    --                                            G     210   369 0.48 0.026                                                                              --    >5000                                         __________________________________________________________________________     .sup.a Medullary pig kidney tissue,                                           .sup.b Rat liver membrane,                                                    .sup.c Rat aortic ring tissue,                                                .sup.d Rat uterus,                                                            .sup.e Hydropenic rat,                                                        .sup.f estimated; compound noncompetitive.                                    ##STR3##                                                                       -                                                                            ##STR4##                                                                     - -                                                                            ##STR5##                                                                     - -                                                                            ##STR6##                                                                     - -                                                                            ##STR7##                                                                     - -                                                                            ##STR8##                                                                     - -                                                                            ##STR9##                                                                 

The data in Table 1 demonstrate a shift to potent V₁ and OXTantagonistic activity from the potent V₂ -receptor antagonismrepresented by a representative Pmp¹ compound. Some V₂ -antagonism ishowever still demonstrated by the compounds of this invention.

The following examples are intended solely to teach the preparation ofthe compounds of this invention. All temperatures are in degreesCentigrade.

EXAMPLE 1 Solid Phase Synthesis of Supported Linear Peptide-BHA ResinStarting Material

For the solid phase synthesis of the titled resin-supported peptides, a7 or 8-position unit-resin material, for example, Boc. Arg(Tos)BHA resin(1.00 mmol/g of resin), was used as a starting material. It was preparedby reacting Boc-Amino Acid-(Tos if necessary), 3 mmol, with thebenzhydrylamine resin, 1.0 mmol, in dimethylformamide for two hours. Thebenzhydrylamine resin as a free base was swelled in methylene chlorideovernight. It was washed once with 7% diisopropylethylamine (DIEA) inmethylene chloride, then 6×1 min. with methylene chloride, and finally2×1 min. with predried dimethylformamide. The loading of BOC-amino acidon the resin was carried out twice on the shaker using1-hydroxybenzotriazole (HBT, 6 mmol), and dicyclohexylcarbodiimide (DCC,3 mmol). A quantitative ninhydrin test and amino acid analysis wereperformed routinely after loading to determine the percentage loading onthe resin.

The appropriately protected amino acids were coupled sequentially on theBoc-amino acid-resin using the Beckman peptide synthesizer 990-B or ashaker. The program used for each coupling, except Boc-Asn andMpa(4-MeBzl), was as follows:

(1) Washed with methylene chloride (3 times, 1 min).

(2) Prewashed with 50% trifluoroacetic acid in methylene chloride (1time, 1 min).

(3) Deprotection with 50% trifluoroacetic acid in methylene chloride (20min).

(4) Washed with methylene chloride (3 times, 1 min).

(5) Prewashed with 7% DIEA in methylene chloride (1 time, 1 min).

(6) Neutralized with 7% DIEA in methylene chloride (1 time, 10 min).

(7) Washed with methylene chloride (3 times, 1 min).

(8) Protected amino acid (3 mmol) in methylene chloride, followed byaddition of DCC, 3 mmol, 10 ml of 0.3M in methylene chloride, andcoupling for two hours.

(9) Washing with methylene chloride (3 times, 1 min).

(10) Washing with ethanol/methylene chloride (1:1) (3 times, 1 min).

(11) Washing with methylene chloride (3 times, 1 min).

In case of coupling of Asn moiety, 1-hydroxybenzotriazole (HBT, 6 mmol)was used, 10 ml of 0.6M in dimethylformamide. Dry dimethylformamide wasalso used as solvent when Mpa(4-MeBzl) was coupled onto the peptideresin, using 4-dimethylaminopyridine (DAP, 3 mmol). Completion of eachcoupling reaction was monitored by the ninhydrin test. Thep-methylbenzyl group was used to protect the thiol groups of Cys and theβ-mercaptopropionic acid (Mpa) moieties.

The benzhydrylamine resin was analyzed by nitrogen analysis to fallwithin 0.72-1.03 mmol per 1 gram. Each protected amino acid unit waspurchased from commercial sources or synthesized by known procedures.Successful coupling incorporated 0.4 to 0.732 mmole per gram of thefirst amino acid.

EXAMPLE 2 ##STR10##

The protected peptide intermediate resin,dPen(4-MeBz)-Tyr(Me)-Phe-Gln-Asn-Cys(4-MeBz)-Arg(Tos)-BHA resin (1.85 g,obtained from 1.0 mmol/g amine/resin (N₂ -analysis) using a Beckmanpeptide synthesizer, 990-B), was reacted with anhydrous hydrogenfluoride (30 ml) in the presence of 3.0 ml of anisole at 0° for 50 min.After evaporation in vacuo to dryness, the residue was treated withanhydrous diethyl ether. The crude peptide was extracted withdimethylformamide (90 ml) and 33% acetic acid (90 ml) into 3.5 l ofde-aerated water previously adjusted to pH 4.5. The aqueous diluteddisulfhydryl octapeptide was cyclized using 0.01M potassium ferricyanidesolution at pH 7.12 until color persisted for 30 minutes. After thecompletion of the oxidation reaction, the pH of the solution wasadjusted to 4.5 using glacial acetic acid. This solution was passedthrough a weakly acid acrylic resin (Bio-Rex 70) column (2.5×12 cm)slowly. The column was eluted with pyridine-acetate buffer (30:4:66,pyridine/glacial acetic acid/water). The pyridine acetate solution wasthen removed by distillation in vacuo. The residue was lyophilized from5% acetic acid to give 610 mg (60%) of crude titled peptide.

Purification

(1) Counter-current distribution (CCD): Sample 610 mg, n-BuOH/HOAc/H₂ O,(4:1:5), 240 transfers. Tubes 106-148 gave 293 mg of product.

(2) Gel Filtration, Sephadex-G-15, 0.2M HOAc: 100 mg gave fraction a, 46mg, and fraction b, 23 mg of pure material.

Physical Data

C₄₄ H₆₃ N₁₃ O₁₁ S₂ ; molecular weight, 1013.393

Fast atom bombardment, mass spectra (FAB): (M═H).sup.⊕ 1014, (M-H).sup.⊖1012

Amino acid Analysis (AAA): Asp (1.00), Glu (1.19), Gly (1.10), Cys(0.45), Tyr (0.60), Phe (1.09), Arg (0.97)

Peptide Content: 63.4% (amino acid content), 84.93% (N₂ -analysis).

Chromatography

1. Thin layer chromatography (TLC)

(a) B/A/W/E (1:1:1:1), R_(f) =0.66

(b) B/A/W/P (15:3:3:10), R_(f) =0.34

2. High pressure liquid chromatography (HPLC) "Altex" ultrasphere ODScolumn, 5μ, 4.5 mm×25 cm 0.1% TFA a, CH₃ CN b:

(a) Gradient, 80 a/20 b to 50:50, a/b, in 15 min.

k'=4.72

(b) Isocratic, 70 a:30 b

k'=2.6.

EXAMPLE 3 ##STR11##

The protected peptide-resin,dPen(4-MeBzl)-Tyr(Me)-Phe-Gln-Asn-Cys(4-MeBzl)-Arg(Tos)-BHA-resin (2.0g, obtained from 1.0 mmol/g of amine/resin (N₂ -analysis), 0.732 mmol/gincorporation of Boc-Arg(Tos) (AAA), using the manual shaker), wasreacted with 30 ml of anhydrous hydrogen fluoride in the presence of 3.0ml of anisole at 0° for 50 minutes. After evaporation in vacuo todryness, the residue was treated with anhydrous diethyl ether. The crudepeptide was extracted with dimethylformamide (75 ml), and 33% aceticacid (75 ml) and taken into 3.5 liter of de-aerated water which had beenpreviously adjusted to pH 4.5. The aqueous diluted disulfhydrylheptapeptide was oxidatively cyclized using 0.01M potassium ferricyanidesolution at pH 7.12 until color persisted for 30 minutes. After thecompletion of the oxidation reaction, the pH of the solution wasadjusted to 4.5 using glacial acetic acid. This solution was passedthrough a weakly acid acrylic resin (Bio-Rex-70) column (2.5×10 cm)slowly. The column was eluted with pyridine-acetate buffer (30:4:66),pyr(HOAc/H₂ O). The pyridine acetate solution was removed in vacuo. Theresidue was lyophilized from 10% acetic acid to give 525 mg (75%) ofcrude titled peptide.

Purification

(1) Counter-current distribution (CCD): Sample 525 mg, n-BuOH/HOAc/H₂ O(4:1:5), 240 transfers

(a) Tubes 128-152, 121.3 mg

(b) Tubes 118-127+153-170, 118 mg pure

(2) Gel Filtration, "Sephadex" G-15, 0.2M HOAc: 89.66 mg from 1a,obtained (a) Fr, 42 mg, (b) Fr, 30.5 mg pure

Physical Data

C₄₂ H₆₀ N₁₂ O₁₀ S₂ ; molecular weight, 956.371

FAB: (M+H).sup.⊕ 957, (M-H).sup.⊖ 955

AAA: Asp (1.00), Glu (1.07), Cys (0.48), Tyr (0.57), Phe (0.99), Arg(0.89)

Peptide Content: 72.2% (AAA), 82.54% (N₂ -analysis).

Chromatography

1. TLC; B/A/W/E (1:1:1:1), R_(f) =0.76

2. HPLC; "Altex" ultrasphere ODS column, 5μ, 4.5 mm×25 cm (a) 0.1% TFA(b) CH₃ CN

(a) Gradient; 80 a/20 b to 50 a/50 b in 15 min,

k'=5.67

(b) Isocratic; 70 a:30 b,

k'=2.93

EXAMPLE 4 ##STR12##

The protected peptide resin,d-Pen(4-MeBzl)-D-Tyr-(Et)-Phe-Gln-Asn-Cys(4-MeBzl)-Arg(Tos)-BHA-resin(2.56 g which was obtained from 0.72 mmol/g, amine/resin (N₂ -analysis),0.4 mmol/g incorporation of Boc-Arg(Tos) (AAA) using an automatedpeptide synthesizer, Beckman 990-B), was reacted with 30 ml of anhydroushydrogen fluoride in the presence of 3-4 ml of anisole at 0° for onehour. After evaporation of excess hydrogen fluoride in vacuo to dryness,the residue was treated with anhydrous diethyl ether. The crude peptidewas extracted with dimethyl formamide (75 ml) and 40% acetic acid (75ml) and taken into 3.5 l of deaerated water which had been previouslyadjusted to pH 4.5. The aqueous diluted disulfhydryl heptapeptide wasoxidatively cyclized using 0.01M K₃ Fe(CN)₆ solution at pH 7.17 untilcolor persisted for 30 minutes. After the completion of the oxidationreaction, the pH of the solution was adjusted to 4.5 using glacialacetic acid. This solution was passed through a weakly acid acrylicresin (Bio-Rex 70 ) column (2.5×10 cm) slowly. The column was elutedwith pyridine-acetate buffer (30:4:6, pyr/HOAc/H₂ O). Thepyridine-acetate solution was removed in vacuo, and the residue waslyophilized from 10% acetic acid to give 450 mg (83.85%) of the crudetitled peptide.

Purification

1. Counter-current distribution (CCD): Sample 450 mg, n-BuOH/HOAc/H₂ O,(4:1:5), 240 transfers

a. Tubes 154-182, 110.70 mg

b. Tubes 138-153, 193.44 mg

c. Tubes 132-137+183-190, 46.98 mg

2. Polyacrylamide resin ("Biogel" P2) column, 0.2M HOAc used 93 mg from1a obtained (a) Fr 50.71 mg, (b) Fr 27.42 mg pure.

Physical data

C₄₃ H₆₂ N₁₂ O₁₀ S₂ ; molecular weight, 970.372

FAB: (M+H).sup.⊕ 971, (M-H).sup.⊖ 969

AAA: Asp (1.00), Gln (0.94), Cys (0.31), Tyr (0.62), Phe (1.00), Arg(0.97)

Chromatography

1. TLC;

(a) B/A/W/E (1:1:1:1), R_(f) =0.657

(b) B/A/W (4:1:5 upper), R_(f) =0.37

2. HPLC; "Altex" Ultrasphere ODS column, 5μ, 4.5 mm×25 cm

(a) Gradient; 80 a/20 b to 50 a/50 b in 20 min

k'=9.01

(b) Isocratic; 70 a/30 b

k'=2.46

EXAMPLE 5 ##STR13##

The protected peptide-resin,Mpr(4-MeBzl)-D-Tyr-(Et)-Ile-Thr(OBzl)-Asn-Cys(4-MeBzl)-Arg(Tos)-Gly-BHA-R(2.33 g, obtained from 0.72 mmol/g amine/resin (N₂ -analysis), 0.46 mmolincorporation of Boc-Gly (AAA), using an automated peptide synthesizer,Beckman 990-B), was cleaved, deprotected by hydrogen fluoride andoxidatively cyclized, as described above, to give crude titled peptide,500 mg (87.81%).

Purification

(1) CCD: Sample 500 mg using BuOH/HOAc/H₂ O (4:1:5), 240 transfers

(a) Tubes 138-166, 112.95 mg pure

(b) Tubes 126-137+167-180, 161.59 mg

(c) Tubes 122-125+181-190, 87.20 mg

(2) HPLC:

Sample, 53.9 mg (from 1a), "Altex" ODS, 10 mm×25 cm, 5u, flow rate 5ml/min, 0.1% TFA/CH₃ CN (60/40), isocratic 220 nm (2.0 AUFS), injection7.19 mg/400 ml to give 45 mg of pure sample of the titled peptide.

Physical data

C₃₉ H₆₂ N₁₂ O₁₁ S₂ ; molecular weight, 938.375

FAB: (M+H).sup.⊕ 939, (M-H).sup.⊖ 937

AAA: Asp (1.00), Thr (0.75), Gly (0.93), Cys (1.00), Ile (0.79), Tyr(0.55), Arg (0.88).

Peptide Content: 68.7% (AAA); 77.78% (N₂ -analysis)

Chromatography

(1) TLC;

(a) B/A/W/E (1:1:1:1), R_(f) =0.69

(b) B/A/W (4:1:5 upper), R_(f) =0.446

(2) HPLC; a 0.1% TFA b CH₃ CN

(a) Gradient 80 a/20 b to 50 a/50 b in 15 minutes

k'=3.6

(b) Isocratic 65 a/35 g

k'=1.56

EXAMPLE 6 ##STR14##

The protected peptide resin,Mpr(4-MeBzl)-D-Tyr-(Et)-Ile-Thr(OBzl)-Asn-Cys(4-MeBzl)-Arg(Tos)-BHA-R(2.2 g, obtained from 1.0 mmol/g amine/resin (N₂ -analysis), 0.485mmol/g incorporation of Boc-Arg(Tos), (AAA), using the manual shaker),was reacted with hydrogen fluoride and oxidized as described previouslyto give crude titled peptide, 245 mg (57.2%).

Purification

(1) CCD; n-BuOH/HOAc/H₂ O (4:1:5), 240 transfers

(a) Tubes 162-190, 156.75 mg

(b) Tubes 156-161+191-200, 56.43 mg

Physical data

C₃₇ H₅₉ N₁₁ O₁₀ S₂ ; molecular weight, 881.353

FAB: (M+H).sup.⊕ 882, (M-H).sup.β 880

AAA: Asp (1.00), Thr (0.85), Cys (0.89), Ile (0.75), Tyr (0.52), Arg(1.27)

Peptide Content: 72.2% (AAA) and 85.58% (N₂ analysis).

Chromatography

(1) TLC;

(a) B/A/W/E 1:1:1:1), R_(f) =0.63

(b) B/A/W/P (15:3:3:10), R_(f) =0.66

(2) HPLC; a 0.1% TFA b CH₃ CN

(a) Gradient 80 a/20 b to 50 a/50 b in 20 min

k'=5.34

(b) Isocratic 65 a:35 b

K'=2.0

EXAMPLE 7 Procedure for the general synthesis of the acid end products(I, A is OH) or cyclic acid intermediates (I, A is OH and Z may be asingle bond)

Boc-AA-Merrifield resin is made by coupling a Boc-AA to Merrifield resinusing the known cesium salt method to give Boc-AA-OCH₂ C₆ H₄ -resinwhich is used as the starting material for the synthesis. The synthesisis carried out on the Beckman 990-B peptide synthesizer using thefollowing protocol. Three equivalents of each of the amino acids aredissolved in their appropriate solvents [for example, the Bocderivatives of 4-MeBzl-Cys, Val, Phe and S-4-MeBzl-Mpa in methylenechloride, Asn in dimethylformamide, D-Tyr(Et) or BrZ-D-Tyr in 1:1methylene chloride/dimethylformamide] and are coupled using an equimolaramount of dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole(HBT) except for the coupling of 4-MeBzl-Mpa where 1.0 equivalent ofdimethylaminopyridine is used as catalyst. The extent of coupling isdermined by qualitative ninhydrin analyses of each aliquot sample andcouplings are repeated when necessary. The Boc groups are removed using1:1 trifluoroacetic acid/methylene chloride and, after washing, the freeamine is generated using 5% diisopropylethylamine/methylene chloride.The sequence of the peptide is checked using solid phase sequencingbefore coupling of the 4-MeBzl-Mpa and its homogeneity confirmed. Afterthe final coupling, the peptide is dried to give the peptide-resin.

1.1 Grams (0.5 mmole) of the peptide resin with 3 ml of anisole isstirred 60 min at 0° (ice bath) in 25 ml of anhydrous liquid hydrogenfluoride (HF). The HF is, then, removed under reduced pressure at 0°.The residue is washed with ethyl ether and the peptide eluted withdimethylformamide, 20% acetic acid and 0.3N ammonium hydroxide.

The filtrate is added to 2 l of degassed water and the pH adjusted to7.1 with conc. ammonium hydroxide. A 0.01M solution of potassiumferricyanide is then added dropwise with stirring until a faint yellowcolor persisted (about 41 ml).

The resulted solution is then passed through a flash column (5 cm×15 cm)of a packing of silica gel coated with a C-18 silane. The column is,then, washed with 350 ml of water and the peptide eluted with 500 ml of1:1 acetonitrile/water (0.25% trifluoroacetic acid) in 20 ml fractions.

Product containing fractions (TLC) are combined and concentrated. Theresidue is dissolved in conc. acetic acid, diluted with water andlyophilized to yield the acid peptide. The Cys(OH) or Z(OH)intermediates are used, without further purification, for the synthesisof the end product peptides.

[1-dPen-2-Tyr(Me)-4-Gln-7-desPro-8-des-Arg-9-desGly(NH₂)] vasopressin(0.3 mmol, prepared as described above) and Arg(NH₂) (0.9 mmol) arereacted in dimethylformamide in the presence of dicyclohexylcarbodiimide(0.9 mmol) and 1-hydroxybenzotriazole (1.8 mmol) at 0°-20° for 6 hours.The volatiles are removed under vacuum. The residue is purified as inExample 2 to give[1-dPen-2-Tyr(Me)-4-Gln-7-desPro-7-Arg(NH₂)-8-desArg-9-desGly(NH.sub.2)]vasopressin.

Reaction of the above Cys(OH) peptide with Arg-Gly(NH₂) under similarreaction conditions gives the corresponding dipeptide-tailed vasopressinderivative.

EXAMPLE 8

Substituting Bos-Lys(ClZ) in Example 2 for the arginine unit gives##STR15##

Substituting Boc-Orn(ClZ) in Example 2 for the arginine unit gives##STR16##

Substituting Boc-Abu in Example 3 for the Gln unit gives ##STR17##

Substituting Boc-Leu for the D-Tyr(Et) unit and D-Arg for the arginineunit in Example 5 gives ##STR18##

Substituting Boc-Chg for the threonine unit in Example 6 gives ##STR19##

Substituting Boc-Tyr(BrZ) in Example 6 for the D-Try(Et) unit in Example6 gives ##STR20##

Substituting Boc-α-aminophenylbutyric acid for the Tyr(Me) unit ofExample 3 gives ##STR21##

Other representative compounds which are prepared by the methods ofsynthesis described above are: ##STR22##

EXAMPLE 9 Parenteral Dosage Unit Compositions

A preparation which contains 10 mcg of the cyclic peptide of Example 2as a sterile dry powder for parenteral injection is prepared as follows:10 mcg of the peptide amide is dissolved in 1 ml of an aqueous solutionof 20 mg of mannitol. The solution is filtered under sterile conditionsinto a 2 ml ampoule and lyophylized. The powder is reconstituted beforeeither intramuscular or intravenous injection to a subject sufferingfrom hypertesion or shock susceptible to anti-ADH mechanism of action.The injection is repeated as necessary, from 1-5 times daily or incontinuous i.v. drug inmjection. Other desPropeptides of this inventionare made up and used in like manner.

Nasal Dosage Unit Compositions

20 Milligrams of finely ground desPropeptide of this invention such asthe product of Example 2 is suspended in a mixture of 75 mg of benzylalcohol and 1.395 g of a suspending agent such as a commercial mixtureof semi-synthetic glycerides of higher fatty acids. The suspension isplaced in an aerosol 10 ml container which is closed with a meteringvalve and charged with aerosol propellants. The contents comprise 100unit doses which are administered intranasally to an edematous subjectfrom 1-6 times a day.

What is claimed is:
 1. A polypeptide compound having the formula:##STR23## in which: P is Phe, Ile, Phe(4'-Alk), Tyr or Tyr(Alk);X is a Dor L isomer of Val, Nva, Leu, Ile, Pba, Phe, Phe(4'-Alk), Trp, Nle, Cha,Abu, Met, Chg, Tyr or Tyr(Alk); Y is Val, Ile, Abu, Ala, Chg, Gln, Lys,Cha, Thr, Nle, Phe, Leu or Gly; Z is a D or L isomer of Arg, Harg, Leu,Lys or Orn; A is Gly(NH₂), Gly, Gly(NH-Alk), OH, NH₂ or NHAlk; and R is,each, hydrogen or methyl, each of said Alk groups being a lower alkyl of1-4 carbons, or a pharmaceutically acceptable salt thereof.
 2. Acompound of claim 1 in which each R is methyl.
 3. A compound of claim 1in which each R is hydrogen.
 4. A compound of claim 1 in which X is a Dor L isomer of Tyr or Tyr(Alk).
 5. A compound of claim 1 in which Y isThr.
 6. A compound of claim 1 in which Y is Gln.
 7. A compound of claim1 being[1-deaminopenicillamine-2-(O-methyl)tyrosine-7-desproline-8-arginine-9-desglycine]vasopressinhaving the structural formula: ##STR24##
 8. A compound of claim 1 being[1-deaminopenicillamine-2-(O-methyl)tyrosine-7-desproline-8-arginine]-vasopressinhaving the structural formula: ##STR25##
 9. A compound of claim 1 being[1-deaminopenicillamine-2-D-(O-ethyl)tyrosine-7-desproline-8-arginine-9-desglycine]vasopressinhaving the structural formula: ##STR26##
 10. A compound of claim 1 being[1-β-mercaptopropionicacid-2-D-(O-ethyl)tyrosine-3-isoleucine-4-threonine-7-desproline-8-arginine-9-desglycine]-vasopressinhaving the structural formula: ##STR27##
 11. A compound of claim 1 being[1-β-mercaptopropionicacid-2-D-(O-ethyl)tyrosine-3-isoleucine-4-threonine-7-desproline-8-arginine]vasopressinhaving the structural formula: ##STR28##